Relevance: Abused drugs stimulate reward pathways in the brain that are implicated in the development of addiction. These reward pathways are activated by the neurotransmitter dopamine in the striatum, which functions by activating G-proteins. G-proteins act as a molecular switch; they are turned "on" by dopamine receptors, and turned "off" by a class of proteins called Regulator of G-protein Signaling, or RGS proteins. A striatum-specific RGS protein, RGS9-2, has been shown to reduce the reward effects and behavioral responses of the dopamine pathway activator cocaine. This proposal seeks to define the mechanism of RGS9-2 inhibition of dopamine signaling in response to addictive drugs like cocaine. Project summary: Dopamine signaling in the ventral striatum plays a critical role in addiction to multiple agents. The ability of a novel striatal-specific Regulator of G-protein Signaling (RGS) protein, RGS9-2, to regulate dopamine signaling in vivo has made it an attractive target for the development of addiction therapeutics. Although the mechanism by which the RGS domain of RGS9-2 may deactivate G-proteins downstream of dopamine receptors is well defined, RGS9-2 contains three additional domains, each of which has been implicated in interactions with other cellular signaling components. Further, multiple receptor pathways exist in striatum that are differentially regulated by RGS9-2. The first goal of this proposal is to define RGS-independent mechanisms of RGS9-2 regulation of dopamine signaling and determine the contribution of each functional domain in the overall cellular function of RGS9-2. The ability of the RGS9-2 G-gamma-like (GGL) domain to form novel G-protein heterotrimers will be determined biochemically using nucleotide exchange assays. The relevance of these GGL-mediated functions and other RGS independent functions to RGS9-2 activity will be determined in cell signaling assays using truncated and mutated RGS9-2 constructs that lack defined domains. Finally, the receptor-selectivity of RGS9-2 regulation among striatal receptors will also be determined in cell signaling assays. The mechanism for specificity will be investigated using steady state GTPase assays to define the role of G-protein specificity and immunoprecipitation and confocal microscopy to define the role of receptor-RGS protein interactions. [unreadable] [unreadable]